Jan 09, 2026
Synaptic vesicle preparation from mouse brain
- Akio Mori1,
- Robert Edwards1
- 1University of California San Francisco
Protocol Citation: Akio Mori, Robert Edwards 2026. Synaptic vesicle preparation from mouse brain. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyqndg5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 19, 2025
Last Modified: January 12, 2026
Protocol Integer ID: 233001
Keywords: synaptic vesicle preparation from mouse brain, purification of synaptic vesicle, synaptic vesicle preparation, synaptic vesicle, brains of mice, mouse brain, glycerol gradient centrifugation, brain tissue, purification, gradient centrifugation
Abstract
This protocol describes the preparation and purification of synaptic vesicles (SVs) from the brains of mice. Brain tissue is homogenized in sucrose-based SHT buffer, followed by sequential centrifugation steps to obtain the LP2 fraction. Synaptic vesicles are subsequently purified by glycerol gradient centrifugation, and SV-enriched fractions are collected for downstream biochemical analyses.
Materials
- Sucrose (≥99.5%) (Sigma-Aldrich)
- Mg-EGTA
- HEPES-Tris, pH 7.4
- cOmplete‱ Protease Inhibitor Cocktail (11836153001, Roche)
- EGTA
- MgCl₂
- Glycerol (molecular biology grade)
Buffers
- SHT buffer: 0.3 M sucrose, 1 mM Mg-EGTA, 10 mM HEPES-Tris, pH 7.4
- Osmotic lysis buffer: 8 mM HEPES-Tris, pH 7.4
- Glycerol gradient buffer (5–25%): 150 mM NaCl, 10 mM HEPES-Tris (pH 7.4), 1 mM EGTA, 0.1 mM MgCl₂, 5–25% (v/v) glycerol (formed as a continuous gradient)
Troubleshooting
Tissue homogenization
Dissect mouse brains and place them in ice-cold SHT buffer supplemented with protease inhibitor.
Homogenize the tissue in 10 mL SHT buffer using a glass–Teflon homogenizer ( 9 strokes at 900 rpm).
Initial fractionation
Centrifuge homogenate at 871 × g, 10 min, 4°C. Collect the supernatant (S1).
Centrifuge S1 at 12,000 × g, 15 min, 4°C to obtain the P2 pellet (crude synaptosomes).
Synaptosome washing and osmotic lysis
Resuspend the P2 pellet in SHT buffer and centrifuge at 14,500 × g, 15 min, 4°C. Discard the supernatant
Resuspend the pellet in 8 mM HEPES-Tris (pH 7.4) and incubate on ice for 45 min to induce osmotic lysis.
Homogenize briefly to complete disruption using a glass–Teflon homogenizer (five strokes at 3,000 rpm) .
Preparation of LP2 fraction
Centrifuge the lysate at 32,500 × g, 20 min, 4°C to obtain supernatant LS1.
Centrifuge LS1 at 280,000 × g, 2 h, 4°C to obtain the LP2 pellet.
Resuspend the LP2 pellet in SHT buffer diluted to 40 mM sucrose (prepared by diluting standard SHT buffer: 0.3 M sucrose, 1 mM Mg-EGTA, 10 mM HEPES-Tris, pH 7.4).
Resuspention
a. Use a 200 μL pipette tip cut at ~1/4 from the distal end and pipette up and down 100 times.
b. Switch to a 200 μL tip cut at ~1/8 from the distal end and pipette 100 times.
c. Finally, use a standard (uncut) 200 μL tip and pipette 100 times to complete resuspension.
Aliquot the resuspended LP2, flash-freeze in liquid nitrogen, and store at –80°C.
Glycerol gradient purification
Prepare a 5–25% glycerol gradient using the glycerol gradient buffer using a gradient maker to form a continuous gradient.
Layer the LP2 sample onto the gradient.
Centrifuge at 270,000 × g for 1 h 4 min at 4°C.
Collect 0.5 mL fractions from the top using a gradient fractionator or connected fraction collector, ensuring smooth and continuous recovery of the gradient.
Use fractions 5–12, which usually contain purified synaptic vesicles, for subsequent experiments.